Method of preparing pteridines



Patented July 11,1951 2 550 1 wads E PREPARING David I. Weisblat andArthur, R. Hanze, Kala;

' 'mazoo, Michl, 'ass'ignors'to The Upjohn (fo n e a-am e iee eii e 9!.

No Drawing. ApplicationJuly 31, 194;}, Serial No.

9 claims. (Q1, zen-251.5) 1 2 This invention relates to a method forpreparlsame configuration as 'l(+)-glutamic acid aping N-((2-amino-4hydroxy-6 pteridyl1methyD pears to bei denti cal with the so-called Lf'csei p-aminobenzoate compounds, part1cularlyto a factor 'orvitamin'Bc'isolated'from'liver. Other method for preparing suchcompounds related compounds of the same general nature, but havcloselyto the group of substances commonly reing two or more glutamic acidresidues connected ferred' to as folic acidfs and to intermediatesthrough the gamma carboxyl groups which can useful in the method. p beprepared by the method given, appear to be Compounds which can beprepared by the identical with and to have the same biological method ofthe invention are. the N-p((2amino-4} activity of still other, membersof, the folic acid yd i yr -pto dyl)methyl)' e p aminobenzoat'e 10group. The value of methods for preparing these tompounds having thegeneral formula i and related compounds synthetically 'is' apparent.

The reactions involved in the method of the invention are indicated inthe, accompanying di- N N CH2 coon agram- I i I According to the methodof, the 1nvent1on, the k N/ W E E R QM N-y ((2-amino-4-;hydroxy e 7 pteridyllmethyD- p -a nobenz'oate Com on s areprepa'red by wherein R isfrom the group consisting of hy ng'a nr gture'fcg pr n ZAQSL PIiQmlpQ-G; drogen and the alkyl radicals, and n is from the hydroxypyri'midihe(I), a ino engoalte com: IIQa, ha ketone group consisting of zero andthe positive integers pound having the Formul 1 to 7, inclusive. havingthefforhiula' negn'e an a rt (in e n h ami f mmands of t e n nt T e t st d e i ell vf at f em out when both a glutamic acid residue andfap;amino 40 "C; 'or" lower to about 100* C'. {or several minbenzoic acidresidue: are included in the molecule, utes'to several'hours andthefiworkd up in any the, nitrogen atomofthoglutamic acidresidue is,convenientmanner'torecdvefthdN Z(ZQaininofor convenience,hereinreferredto bythesymb'ol 4-hydroxy 6 preridy1)methy1 -"paminoben- "N" andthe nitrogen atom of the p-amino ioate compound. One convenient methodfor re benzoic acid residue is referred to by the symbol covering theproduct consistsiii dilutingthe mix- N. As indicated by theformulagiven, corn ture witha relatively 'largvolume'of'watenor poundscontaining more than one glutamic acid or aqueous "alcohol and filteringafteradjusting the ester residue contemplat d y the invention are pH ofthe diluted mixture to aboutS." The N-'; those wherein only the gammacarboxyl groups (2-,amino-4-.-hydroxy 6 pteridyl)methyl)'-pare involvedin the peptide linlgages'; aminobenzoate'compound is thus obtained insolid I II III Certain of the N.- ((2-amino-4-hydroxy-6 form and can bepurified further, if desired, by pteridyl) methyl) w p aminobenzoatecompounds repeated washing with cold alcohol. pr p r d by the method ofthe invention appear The reaction is carriedoutconveniently either to beidentical with certain compounds of'the by mixing all of the reactantstogether and heatgroup isolated from natural sources and referred ingthe mixture or, with advantage'in certain into broadly in the art asfolio acids. Thus N'- stances, by adding the haloketone to one of the(N- (2 amino-ei hydroxy d pteridyl)methyl) p other reactants andsubsequently adding the third aminobenzoyl')-glutamic' acid(pteroyl-glutamic reactant to the mixture with or without interacid)wherein the glutamic acid residue has the mediate heating before addingthe third reactant.

Inert diluents which can be used include aqueous acetic acid, propionicacid, ethylene glycol, propylene glycol, the polyglycols and theiresters as well as many others.

It has been noted that advantages results in the way of increased yieldand a faster reaction rate are generally obtained, particularly whenusing a chloroor bromo-ketone, byincluding a small proportion of analkali metal iodide, e. g. of sodium or potassium iodide, in themixture. It is also frequently advantageous, particularly when anaqueous acetic acid medium is used, to

include sodium acetate or other alkaline salt in the mixture to increasethe pH somewhat and bufier the mixture. The reaction is preferably, butnot necessarily, carried out under anhydrous conditions. Although themechanism of the reaction is not completely understood, it is found thatwhen the reaction is carried out as described, substantially completearomatization of the heterocyclic ring occurs with the formation of thepteridine nucleus. N-((2-amino-4-hydroxy- G-pteridyl) methyl)-p-aminobenzoate compounds which are esters can be hydrolyzed readily tothe corresponding acids with dilute alkalies.

The haloketones which can be used in the reaction are the dihaloacetonesand the mono-halomono-hydroxy acetones having the general formulaXCHzCOCH2R wherein X is a halogen from the group consisting of chlorine,bromine and iodine and R is from the group consisting of chlorine,bromine, iodine and the hydroxyl radical.

p-Amino-benzoate compounds having the Formula III which can be used inthe process include those wherein n is zero, i. e. p-aminobenzoic acid,and alkyl esters thereof, and also those wherein n is an integer from 1to 7, inclusive, such as N'-(p-aminobenzoyl) -glutamic acid,N'-(paminobenzoyl) -gamma glutamylglutamic acid, N'-(p-aminobenzoyl)gamma glutamyl gamma-glutamylglutamic acid and their alkyl esters.

The p-aminobenzoate compounds wherein n is an integer from the group 1to '7, inclusive, can be obtained in a number of ways, one of which isdescribed in a concurrently filed copending application Serial No.41,888. According to the method of the copending application, ap-aminobenzoate compound having one glutamic acid residue in themolecule is prepared by first reacting glutamic acid or an alkyl esterthereof with a p-nitrobenzoyl halide. The halides referred to butyl,tert-butyl, hexyl, nonyl and dodecyl esters, can be used with equalfacility, if desired. 7

Although the invention is directed particularly, in case of esters ofthe glutamic acid residues, to alkyl esters, the process of theinvention can also be carried out and corresponding compounds preparedusing other esters, such as the phenyl, tolyl, xylyl, cyclohexyl, benzyland many other aryl, aralkyl or cycloalkyl esters.

A preferred modification of the invention comprises the use in thereaction of dichloroacetone and of a p-aminobenzoate compound having oneglutamic acid or ester residue in the molecule, i. e. ofN-(p-aminobenzoyl) -glutamic acid or its ester, to formN(N-((2-amino-4-hydroxy-6- pteridyl) methyl) p aminobenzoyl) glutamicacid or its ester, although the invention is not so limited.

Compounds similar to or identical with those of the folic acid groupmade by using the intermediates or method of the invention, such aspteroylglutamic acid and pteroyl-gamma-glutamyl-gamma-glutamylglutamicacid, which are of greatest value as measured by their biologicalactivity against Lactobaczllus casez' or Streptococcus jecalis R, arethose having the same configuration as l(+) -glutamic acid. However, theinvention also contemplates compounds having the dextro configuration aswell as racemic mixtures.

Certain advantages of the invention are apparent from the followingexamples which are given by way of illustration only and are not to beconstrued as limiting.

Example 1. Diethyl N (p nitrobenzoyl) lglutamate crude diethyl l(+)-glutamate hydrochloride and benzene removed under reduced pressure.

in this connection are the chlorides and the bromides. AnN'-(p-nitrobenzoyl) -glutamic acid or ester is first obtained which,upon reduction, e. g. with hydrogen using platinum oxide as acatalyst,yields an N'-(p-aminobenzoyl) -glutamic acid or ester. The N(p-aminobenzoyl) -glutamic acid can, if desired, be converted to thecorresponding alkyl esters, e. g. by treatment with an alkanol and anesterification catalyst in known matter, or the esters can be hydrolyzedto the corresponding acids. In similar manner, other paminobenzoatecompounds can be prepared having up to seven glutamic acid residues inthe molecule by starting with the corresponding gamina-glutamylglutamicacid or ester containing the requisite number of peptide linkages.

Although the invention will be described, in the case of esters, withparticular reference to the ethyl esters, it is understood that otheralkyl esters, such as the methyl, propyl, iso-PIOPYL milliliters ofpyridine in 200 milliliters of benzene. The mixture was stirred for fivehours, extracted first with dilute hydrochloric acid and then withaqueous sodium bicarbonate and the The residue consisted of grams of apasty, neutral fraction consisting largely ofdiethy1N-(pnitrobenzoyll-l-glutamate. After recrystallization fromdilute ethanol, the ester melted at 93 to 95 C. It had a specificrotation of (a) =18. The sodium bicarbonate extract upon acidificationyielded 42 grams of p-nitrobenzoic acid.

Example 2. Diethyl N (p aminobenzoyl) l-glatamate Crude diethylN-(p-nitrobenzoyl) -l-glu'tamate prepared as in Example 1 was dissolvedin ethanol and reduced with hydrogen under a pressure of 40 pounds persquare inch using platinum oxide as a catalyst. The mixture was thenfiltered to recover platinum and the ethanol evaporated under reducedpressure. There was thus obtained a 52 per cent yield of diethyl N'-'(p-aminobenzoy1)-l-glutamate melting at to 138 C. Uponrecrystallization from dilute ethanol, the ester melted at 140 to 141 C.and had a specific rotation (-a) =9.5 in 95 per cent ethanol.

Anal. Calcd. for CisHzzOsNzZ C, 59.6; H, 6.9; N, 8.7. Found: C, 59.6; H,6.8; N, 9.0.

Example 3.--N'-(p-nitrobenzoyl) -Z-glutamz'c acid Eighteen and one-halfgrams of p-nitrobenzoyl chloride was added over a period of 0.5 hour toa allowed to stand at room temperature for four and one-half hours tohydrolyze the ethyl ester groups. The pH was then adjusted to 3.5 withacetic acid and the mixture cooled in ice and cetrifuged. The solid waswashed three times with water and once with absolute alcohol. The dryproduct consisting of N '(N-((2-amino-4-hy droxy 6 pteridyl) methyl)p-aminobenzoyD- glutamic acid had an activity against L. casei equal to3.4 percent of that of pure folic acid.

Example 10.-N-(N- (2 amino 4 hydroxy 6- pteridyl) methyl) paminobenzoyl) -glutamic acid A solution of '76 milligrams ofl-chloro-3-hydroxypropanone-Z-in l5 milliliters of glacial acetic acidwas added in an atmosphere of nitrogen to a mixture of 99 milligrams of2,4,5-triamino-6-hydroxypyrimidine, 451 milligrams of diethylN'-(p-aminobenzoyl) -glutamate and 40 milligrams of potassium iodide. Arapid change in color of the mixture to deep orange-red occurred almostimmediately. The mixture was stirred for seventy minutes under nitrogenat room temperature at the end of which time the color had changed toreddish-brown. The mixture was then heated for ten minutes on the steambath, the color becoming entirely brown. The mixture was thenconcentrated in'vacuo, twenty milliliters of water was added and the pHof the mixture adjusted to 3.0. The solid which separated was removed bycentrifuging and mixed with ten milliliters of normal sodium hydroxidesolution. The mixture was allowed to stand for about sixteen hours inthe refrigerator and then for thirty minutes at room temperature tohydrolyze the ethyl ester groups. The mixture was homogeneous at thispoint. The pH of the mixture was then adjusted to 3.0 with dilutehydrochloric acid and after cooling the mixture for two hours, it wascentrifuged and. the recovered solid product washed twice with water andonce with acetone. The crude N'-(N- ((Z-amino 4 hydroxy 6 pteridyl)methyl) -p-- aminobenzoyD-glutamic acid thus obtained weighed '70milligrams after drying over phos-- phorus pentoxide and had an activityagainst L. casez' equal to 4 per cent of that of pure folic acid.

Example 11.N- (2 amino 4 hydroxy-G-pteridyl) methyl) -p-aminobenzo2'cacid A mixture of 100 milligrams of 2,4,5-triamino G-hydroxy pyrimidine,90 milligrams of dichloroacetone and 15 milliliters of glacial aceticacid was allowed to stand for 70 minutes at room temperature. Fortymilligrams of potassium iodide and 290 milligrams of p-aminobenzoic acidwere then added. The mixture was allowed to stand at room temperaturefor an additional hour and then heated on a steam bath for 15 minutesand finally allowed to stand overnight at room temperature. The aceticacid was then distilled in vacuo, the residue mixed with 15 millilitersof water and the pH of the mixture adjusted to 3.05. The mixture wascooled and centrifuged and the precipitate washed with alcohol anddried. The crude N-((2-amino-4-hydroxy-6- pteridyl) methyl)-p-aminobenzoic acid thus obtained weighed 100 milligrams and had anactivity against L. casei equal to 9.7 per cent that of pure pteroicacid.

Example 12.-N- (2 amino-4 hydroxy- 6 -pteridyl) methyl) -p-aminobenzoz'cacid A mixture of milligrams of dichloroacetone, milligrams ofp-aminobenzoic acid, 40 milligrams of potassium iodide, 60 milligrams ofsodium acetate and 15 milliliters of glacial acetic acid was allowed tostand for 4 hours at room temperature and then heated for fifteenminutes on a steam bath. The mixture turned dark brown during theheating. The mixture was then cooled and milligrams of sodium acetateand 100 milligrams of 2,4,5-triamino-6-hydroxy pyrimidine were added.The mixture was again allowed to stand for 80 minutes at roomtemperature, heated on a steam bath for fifteen minutes and then allowedto stand for about sixteen hours at room temperature. The acetic acidwas distilled in vacuo and the residue stirred thoroughly with 15milliliters of water. The pH of the mixture was adjusted to 3.0 and themixture then centrifuged. The crystals thus obtained were washed withwater and alcohol and dried. There was thus obtained 95 milligrams ofcrude N-((2- amino-e-hydroxy- 6 -pteridyl) methyl) -p-aminobenzoic acidhaving an activity against L. casei equal to 3.6 per cent that of purepteroic acid.

We claim:

1. The method for preparing an N-((2-aminol-hydroxy-fi-pteridyl) methyl)p-aminobenzoate compound having the formula COOR CH1 N \NHOComntuomomcouoa' HzN \N N/ wherein R. is from the group consisting ofhydrogen and the alkyl radicals and n is from the group consisting ofzero and the integer 1, which includes the step of heating a mixturecomprising 2,4,5triamino6-hydroxypyrimidine, a haloketone having theformula XCHzCOCHzR wherein X is a halogen from the group consisting ofchlorine, bromine and iodine and R is from the group consisting ofchlorine, bromine, iodine and the hydroxyl radical, and ap-aminobenzoate compound having the formula COOR HlN-O-oomm momcmo o..oa'

wherein X is from the group consisting of chlorine, bromine and iodineand R is from the group consisting of chlorine, bromine, iodine and thehydroxyl radical, a p-aminobenzoate compound having the formula COORwherein R is from the group consisting of hydrogen and the alkylradicals and n is from the group consisting of zero and the integer 1,and a catalytic amount of potassium iodide; and separating from thereaction mixture a compound having the formula N N coon NHCO(NHOHCH2CH2CO)OR' 3 wherein R and n have the values given.

7. The method of claim 6 wherein the liquid medium is an aqueoussolution of acetic acid and sodium acetate.

8. The method which includes: mixing in a liquid medium a haloketonehaving the formula XCHzCOCHzR wherein X is from the group consisting ofchlorine, bromine and iodine and R is from the group consisting ofchlorine, bromine, iodine and the hydroxyl radical and a p-aminobenzoatecompound having the formula OOOR wherein R is from the group consistingof hydrogen and the alkyl radicals and n is from the group consisting ofzero and the integer 1; subsequently adding2,4,5-triamino-G-hydroxypyrimidine to the mixture; and separating fromthe reaction mixture a compound having the formula COOR' N N CE:

NH-O-O o monomomoo) .oR' HQN N N/ wherein R and n have the values given.

10 9. The method which includes: mixing in a liquid medium2,4,5-triamino-6-hydroxypyrimidine and a haloketone having the formulawherein X is from the group consisting of chlorine, bromine and iodineand R is from the group consisting of chlorine, bromine, iodine and thehydroxyl radical; subsequently adding to the mixture a p-aminobenzoatecompound having the formula COOR mN-goomndncmomo 0),,o R

wherein R is from the group consisting of hydrogen and the alkylradicals and n is from the group consisting of zero and the integer 1;and separating from the reaction mixture a compound having the formulawherein R and n have the values given.

DAVID I. WEISBLAT. ARTHUR R. HANZE.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS Number Name Date 2,443,165 I-Iultquist et a1 June8, 1948 OTHER REFERENCES Lederle Bulletin, 13 (No. 3), 21 (1948).

1. THE METHOD FOR PREPARING AN N-(2-AMINO4-HYDROXY-6-PTERIDYL) METHYL) -P-AMINOBENZOATE COMPOUND HAVING THE FORMULA